Studies outlined in the revised competing renewal are aimed at understanding the physiologic and therapeutic effect of activating ADO A3 receptors (Rs) in experimental models of colitis. Three specific aims are addressed in distal colon, the therapeutic target for the A3R agonist IB-MECA in TNBS colitis. AIM 1: Determine the physiological role of ADO A3Rs in neural reflexes of the rat distal colon. Hypothesis 1.1 is tested that IB-MECA acts at A3Rs on enteric neurons to inhibit propulsion by recording from AH sensory neurons identified morphologically by neurobiotin injection or S descending motor neurons to circular muscle (CM) identified physiologically by retrograde labeling with Oil. A3 modulation in the inhibitory motor limb of reflex is determined by experiments on CM relaxation, VIP release, strain-gauge recording of large amplitude contractions or peristaltic propulsion of a fecal pellet. Endogenous ADO release will be quantitated by mass spectrometry. AIM 2. Determine the role of A3Rs as a therapeutic target in experimental models of colitis. Hypothesis 2.1 is tested that A3Rs mediate the therapeutic effect of IB- MECA in chronic inflammation induced by TNBS in rats. A1 or A3 antagonists will prove A3R involvement. Hypothesis 2.2 is tested that A3Rs confer protection in genomic models of colitis. It is tested using DSS in C57BL/6 A3 KO or FVB/N profilin transctenic mice with 8-fold up-regulation of A3Rs. Transgenic manipulation of A3R may provide unequivocal proof for receptor signaling and A3R-IB-MECA protection in murine colitis. AIM 3. Determine the cellular and intracellular mechanisms involved in the beneficial effects of IB-MECA treatment in TNBS colitis. Hypothesis 3.1 tests if activation of A3Rs provides neuroprotection in TNBS colitis. It is tested by looking at reversal of neural gene dysregulation, sensory AH and EC cell hyperesponsiveness or H202 oxidant-activation of neurons, analysis of eADO and eADO effects at A3Rs. The therapeutic study with oral IB-MECA more closely parallels acute ex-vivo studies and tests its effect once the damage has occurred in TNBS colitis. Hypothesis 3.2 tests if adenosine A3R activation inhibits oxygen free radicals in rat TNBS colitis using EPRI analysis of tissue and neutrophil free radicals, and gene microarray of oxidant and antioxidant genes. These mechanistic studies will provide new insights into A3R regulation as a therapeutic target in experimental models of lBD and may pave the way towards translational studies. [unreadable] [unreadable]